P1432: THE NOVEL DUAL T/B-CELL SIGNALING MOLECULE CONFERS THE GREATER TUMORICIDAL ACTIVITY AND T-CELL PERSISTENCE OF CD19 CHIMERIC ANTIGEN RECEPTOR T-CELL
Authors: W. Khopanlert, N. Jangphattananont, K. Maneechai, S. Ung, P. Viboonjuntra, S. Terakura, J. Julamanee
Journal: HemaSphere (2022)

Background: Chimeric antigen receptor (CAR) T-cell targeting CD19 incorporating either CD28 or 4-1BB co-stimulatory domains have achieved impressive clinical outcomes for adoptive immunotherapy in B-cell malignancies. However, the recurrences after therapies still occur due to the poor CAR-T persistence. Various approaches have been studied to extend the efficacy including signaling domain modification. The nuclear factor-κB (NF-κB) is a critical signaling pathway for CAR-T cell persistence and anti-tumor activity. Interestingly, this downstream signaling cascade exists in both T- and B-cells. Here, we adopted the third generation CD19CAR incorporated dual T- and B-cell costimulatory molecules; CD28 and CD40 (CD19.28.40z), to improve CAR-T cell functions. Aims: To generate the novel dual T/B-cell signaling domain of CD19CAR (CD19.28.40z CAR) enhancing cytotoxicity and persistence. Methods: The dual T/B-cell costimulatory domain; CD28/CD40, was fused into the anti-CD19CAR backbone; CD19scFv-CD28TM-CD3z-tEGFR, to generate the third generation CAR. The CAR gene was cloned into retroviral packaging cells and transduced into primary human CD3 cells. The CAR+-T cells were enriched using biotinylated anti-EGFR mAb and further expanded with anti-CD3/CD28 beads or gamma-irradiated EBV-LCL cells. The CAR+-T cells were then used for downstream experiments compared with control CD19CARs including CD19.28z, CD19.BBz, and CD19.40z CAR-T cells. Results: All CD19CAR genes were successfully transduced into primary CD3+ cells with the transduction efficacy of 40–60% and slightly higher in CD19.28.40z CAR-T cells. We next investigated the downstream T-cell signaling and found the higher baseline pNF-κB and pRelB of CD19.28.40z CAR-T cells compared to the others. The novel CD19.28.40z CAR-T cells exhibited robust T-cell proliferation after stimulation once with gamma-irradiated CD19-expressing K562 cells regardless of IL-2 supplementation. In terms of cytokine secretion, we observed similar level of IL-2, TNF-α, and IFN-γ concentrations among the CAR constructs after stimulated with target cells. To assess the long-term cytotoxicity, we performed co-culture assay and found that CD19.28.40z CAR-T cells effectively eradicated tumor cell growth throughout the end of the experiment at an effector to target cell ratio of 1:16, compared to control CAR T-cells (Figure 1). Notably, CD19.28.40z CAR-T cells sustained greater proliferation after repetitive weekly stimulation without influencing T-cell fate and exhaustion (Figure 2). The preliminary in vivo murine experiment demonstrated that CD19.28.40z CAR-T cells profoundly suppressed the tumor cell growth in Nalm-6-inoculated NSG mice compared to other control T-cells. Image:Summary/Conclusion: The novel CD28/CD40 signaling domain enhances CD19CAR-T cell proliferative capacity and persistence which translated into the greater anti-tumor efficacy both in vitro and xenograft model.